The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of a DNA sequence of interest (such as DNA insert containing a natural or synthetic gene or gene fragment) into a pre-assembled cloning vector. The cloning vector includes all of the necessary components for replication of the DNA insert in a compatible host cell, e.g., promoter sequence, origin of replication sequence, termination sequence, and a selectable marker sequence. The DNA insert sequences can be derived from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA sequences. Alternatively, the DNA insert sequences can be created synthetically.
Insertion of the DNA sequence of interest can be accomplished by a number of techniques. The most common technique involves restriction enzymes. A restriction enzyme recognition site that is present in both the DNA insert and the vector of interest is cleaved with a restriction enzyme to provide for appropriate termini, the termini of either the DNA insert or the vector are treated with alkaline phosphatase to remove terminal phosphates and avoid undesirable joining, and the DNA sequence of interest is inserted into the vector at the compatible sites during a ligation reaction. A restriction enzyme site present in a pre-assembled vector must be compatible with a restriction enzyme site in the DNA sequence of interest.
Alternatively, the DNA of interest can be modified to obtain compatible restriction sites by filling in of cohesive ends as appropriate, or by the ligation of an appropriate oligonucleotide linker, which can be subsequently cleaved by the restriction enzyme of interest.
Conventional cloning methods can be time consuming and often involve multiple sub cloning steps. Therefore, a need exists for developing a simple and rapid method for synthesizing and identifying an optimal construct for use in a particular application.
This invention pertains to methods for preparing multicomponent nucleic acid constructs. The method of the invention has a wide variety of applications for the expression of synthetic and naturally occurring genes or gene fragments. The invention provides integral vector elements which may be specifically selected or which may varied so as to create a collection of vectors from which optimal configurations can be selected or screened for. These integral vector elements include both vector backbone elements, which do not directly affect the expression or form of the insert gene or gene fragment, and insert modifying vector elements which alter the expression and/or form of the insert-encoded gene product. This system for the rapid and flexible assembly of specific multicomponent nucleic acid constructs is referred to as GEOS (for Genetic Engineering Operating System). GEOS methodology has numerous applications ranging from the assembly of simple circular expression vectors to the production of complex linar assemblies which function as small chromosomes. Various applications of GEOS methodology are discussed in detail below and still others will be apparent to the skilled artisan.
The invention further provides a method of linking the vector element nucleic acid components in a predetermined order so as to produce a nucleic acid multicomponent construct.
In certain preferred embodiments, the GEOS method comprises:
(a) providing the nucleic acid components to be assembled into the construct, each nucleic acid component comprising a double stranded nucleic acid molecule having at least one single stranded 5xe2x80x2 or 3xe2x80x2 terminal sequence, the terminal sequence having sufficient complementarity to a terminal sequence in a separate nucleic acid component so as to allow for specific annealing of complementary sequences and linkage of the components in a predetermined order;
(b) incubating the nucleic acid components under conditions which allow for the specific annealing and linkage of the nucleic acid components to thereby produce the nucleic acid multicomponent construct.
In another preferred embodiments, the GEOS method comprises:
(a) providing the nucleic acid components and one or more linking nucleic acid molecules to be assembled into the construct, each nucleic acid component comprising a double stranded nucleic acid molecule having at least one single stranded 5xe2x80x2 or 3xe2x80x2 terminal sequence, the terminal sequence having sufficient complementarity to a sequence in a linking nucleic acid molecule so as to allow for specific annealing of complementary sequences and linkage of the components in a predetermined order;
(b) incubating the nucleic acid components under conditions which allow for the specific annealing and linkage of the nucleic acid components to thereby produce the nucleic acid multicomponent construct.
The genetic element portion(s) of the nucleic acid components can be double- or single-stranded, though are preferably double-stranded.
In a preferred embodiment of the method, the nucleic acid components are flanked by single stranded terminal sequences and these terminal sequences are preferably non-palindromic. The nucleic acid components can be linked either directly via annealing of 5xe2x80x2 or 3xe2x80x2 complementary terminal sequences or indirectly via a linking nucleic acid molecule (e.g. an oligonucleotide or an adaptor molecule).
The nucleic acid components can be linked either simultaneously or sequentially to form the nucleic acid construct. Sequential assembly is suitable for automation. The method can be used to produce nucleic acid constructs which are functional as assembled or constructs which are used as subcomponents for the assembly of functional constructs.
The method of the invention can be used to synthesize a group of nucleic acid constructs in which one or more of the components can be substituted, in each of the constructs, with a different nucleic acid component, having the same functionality or characteristic utility. This allows for comparison of the different components and production of an optimal construct for a particular application. Toward this end, the nucleic acid components are designed and synthesized in such a way that a group of nucleic acid components belonging in the same category (i.e., having the same functionality or characteristic utility, e.g. a set of nucleic acid components encoding different promoters) possess the same terminal sequences, such that the same category nucleic acid components can be used interchangeably to assemble a nucleic acid multicomponent construct.
The nucleic acid components may also be covalently or non-covalently modified prior to or following assembly of the nucleic acid multicomponent construct. This allows for the synthesis of constructs having biological properties which cannot be obtained easily using current recombinant methods. For instance, the modification utilizes an arylboronic acid reagent, such as phenyldiboronic acid.
The method of this invention is particularly suitable for the construction of nucleic acid vectors. These include plasmid, viral, or phage vectors, or yeast artificial chromosomes. The vector can be a cloning or expression vector and can be used for the expression of cDNA or genomic libraries, genes or gene fragments, mutagenized genes, recombined fusion genes, and artificial genes. The constructs can be employed in prokaryotic, eukaryotic (mammalian or non-mammalian) expression, construction of unique cDNA libraries, protein, antibody and peptide phage display libraries. The constructs can further be employed in gene transfer, gene therapy, and the creation of transgenic organisms.
According to the method, the vector is assembled from nucleic acid components encoding a single functionality or multiple functionalities. In some applications of the invention, more than one biological function may be bundled into a single nucleic acid component. This may be desirable when, for example, one seeks to limit the overall number of components to be assembled into the GEOS construct. In one embodiment of the invention, nucleic acid components encoding an origin of replication, a selectable marker and an insert of interest are used. Depending on the type of vector desired, nucleic acid components encoding other vector functions may also be incorporated (e.g. a promoter, a transcription or translation regulatory element, etc.). An expression vector can be produced using a nucleic acid component encoding a structural gene or gene fragment of interest and additional nucleic acid components encoding regulatory elements required for expression of the gene. For example, a cDNA library expression vector is produced using nucleic acid components encoding a collection of cDNA molecules derived from poly(A)+ mRNA. Importantly, the optimization procedure of interchanging nucleic acid components described above can be used to create an optimal vector for a particular application.
The invention further provides a kit for the production of vectors. In one embodiments, the kit for the production of vectors would minimally comprise nucleic acid components encoding origins of replication and selectable markers and optionally, transcriptional regulatory sequence(s). The kit could also include nucleic acid components encoding other vector functions (e.g. a promoter, a transcription or translation regulatory element, etc.).
The invention further provides a kit for the production of vectors. The kit for the production of vectors would minimally comprise nucleic acid components encoding origins of replication, selectable markers and inserts of interest. The kit could also include nucleic acid components encoding other vector functions (e.g. a promoter, a transcription or translation regulatory element, etc.).
The method of the invention is a highly efficient, rapid, cost effective alternative to current recombinant cloning methods in that it enables users to choose from a broad array of different nucleic acid components or modified nucleic acid components when assembling any construct. The method of the invention allows the rapid construction of customized constructs without the need to use restriction enzymes.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.